In your case I would recommend that you set the Account lookup field in the background (via a before insert trigger as you had suggested) so that the Account field always reflects the correct value. i.e. You don't want to allow users to select the Account lookup value to be anything other than Opportunity's parent Account.

For some reason, if you do infact want users to be able to select the Account value on the custom object and you just want to pre-populate that field with the correct value when the VF page first displays, here's how:

1) Use the following in your custom extension class (assuming 'My_Custom_Obj__c' is the custom junction object between Account and Opportunity)

public with sharing class TestPage2Ext 
{
	My_Custom_Obj__c record {get;set;}

	public TestPage2Ext(ApexPages.StandardController stdController)
	{	
		record = (My_Custom_Obj__c) stdController.getRecord();
		record.account__c = ApexPages.currentPage().getParameters().get('acctId');	
		record.opportunity__c = ApexPages.currentPage().getParameters().get('oppId');
		
	}
	
	public PageReference save()
	{
		insert record;
		return null;
	}
}

2) In your VF page, bind the 'record' variable to the inputFields that accept the value for the Opportunity and Account

<apex:inputField value="{!record.Opportunity__c}"/>
<apex:inputField value="{!record.Account__c}"/>

 3) Add a new custom 'List Button' on the 'My_Custom_Obj__c' custom object. Set the following for the URL of that custom button (assuming that 'TestPage2' is your custom VF page:

/apex/TestPage2?acctId={!Opportunity.AccountId}&oppId={!Opportunity.Id}

 4) In the Page Layout for Opportunity, edit the related list for the 'My_Custom_Obj__c' custom object. Remove the standard 'New' button and instead add the button you created above to the related list.

Now when you users click on this custom button from the related list on an Opportunity record, they'll be taken to a custom VF page with the Opportunity and Account fields pre-populated.

Hope this helps...